Dietary restriction (DR) delays the incidence and decreases the growth of various types of tumors; however, the mechanisms responsible for DR-mediated antitumor effects have not been unequivocally identified. Here, we report that DR suppresses xenograft tumor growth by upregulating a novel signaling pathway. DR led to upregulated aldolase A (ALDOA) expression in xenograft tumors. ALDOA physically interacted with the catalytic subunit of DNA-dependent protein kinase (DNA-PK) and promoted DNA-PK activation. Activated DNA-PK phosphorylated p53 and increased its activity. Although ALDOA can function as an oncogene in cultured cells, it can also activate the tumor suppressor p53. Thus, ALDOA overexpression in the presence of p53 suppressed xenograft tumor growth; however, when p53 was suppressed, ALDOA overexpression promoted xenograft tumor growth. Moreover, we demonstrated that p53 suppression inhibited the antitumor effects of DR. Our results indicate that upregulation of the ALDOA/DNA-PK/p53 pathway is a mechanism accounting for the antitumor effects of DR.